Evolutionary Systematics. 4 2020, 21–33 | DOI 10.3897/evolsyst.4.49537 The phylogeny of Empis and Rhamphomyia (Diptera, Empididae) investigated using UCEs including an over 150 years old museum specimen Caroline Rhodén1, Emma Wahlberg1,2 1 Department of Zoology, Stockholm University, Stockholm, Sweden 2 Department of Zoology, Swedish Museum of Natural History, Stockholm, Sweden http://zoobank.org/58DF97B8-3FE5-4341-86E6-4BD5DDE777E0 Corresponding author: Emma Wahlberg ([email protected]) Academic editor: M. Husemann ♦ Received 19 December 2019 ♦ Accepted 21 January 2020 ♦ Published 4 February 2020 Abstract The genera Empis Linneus, 1758 and Rhamphomyia Meigen, 1822 (Empidoidea, Empididae Latreille, 1809) are two large genera of flies commonly named dagger flies. They are widely distributed in the world with most species described from the Palearctic Region. Empis comprises about 810 described species and Rhamphomyia comprises about 610 described species, together they represent one third of the known species diversity in Empididae. Two recent studies on the phylogeny of the two genera using Sanger sequencing on a few genetic markers, did not support monophyly of them. In this study high throughput sequencing of target enriched molecular data of ultraconserved elements or UCEs was used to investigate the phylogenetic relationships of included representatives of the genera. This method has proven useful on old and dry museum specimens with high amounts of degraded DNA, which was also tested herein. For this purpose, a commercially synthesized bait kit has previously been developed for Diptera which this study was the first one to test. Three out of nine old and dry museum specimens were successfully sequenced, one with an age of at least 154 years. Higher DNA concentration yielded a greater number of reads. Analyses conducted in the study confirmed that bothEmpis and Rhamphomyia are non-monophyletic. Key Words Entomology, high throughput sequencing, next generation sequencing, systematics, taxonomy, target enrichment, UCE Introduction (Cumming 1994; LeBas et al. 2003; Murray et al. 2018). The empidid tribe Empidini Latreille is highly diverse The family Empididae Latreille, in the superfamily Em- and consists of 14 genera spread all over the world, with pidoidea Latreille, commonly known as dagger flies, is a a particular diversity in the Neotropical Region (Wieg- family within Diptera consisting of around 3 051 known mann et al. 2011). The two most species-rich genera in species in the world (Roskov et al. 2019). Dagger flies the tribe are the sister groups of Empis Linneus, 1758 and gets their vernacular name from the long and dagger-like Rhamphomyia Meigen, 1822, and the majority of species piercing mouthparts. An older name is dance flies; how- of these two genera are described from the Palearctic ever, this name is today assigned to the family Hyboti- Region (Watts et al. 2015). Empis constitute about 810 dae Fallén, in the same superfamily. In both Empididae described species and Rhamphomyia about 610 described and Hybotidae many species form swarms where a typ- species according to 2019 Annual Checklist (CoL). To- ical mating ritual, which is perceived as a dance, takes gether they represent more than one third of all known place. Members in the subfamily Empidinae Latreille, Empididae species (Roskov et al. 2019). In an attempt to constitute a high interspecific variation in mating rituals. obtain a better overview of the diversity of the two genera Copyright Caroline Rhodén, Emma Wahlberg. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 22 Caroline Rhodén & Emma Wahlberg: The phylogeny of Empis and Rhamphomyia several subgenera have been established. However, there went well and to validate species determinations in The is no clear number on how many subgenera there are but Barcode of Life Database (BOLD) (Ratnasingham and approximately 24 are given for Empis and 18 for Rham- Hebert 2007). In this study we include both relatively new phomyia (Chvála 1994; Poole and Gentili 1996; Yang et material kept in ethanol sampled between 1995 and 2015 al. 2007; Saigusa 2012; Evenhuis and Pape 2019). and older, dry museum specimens sampled between the Previous studies have indicated that Empis and Rhamph- years 1843 and 1993. The molecular data from the high omyia are non-monophyletic (Watts et al. 2016; Wahlberg throughput sequencing is used to investigate the phyloge- and Johanson 2018). The study by Watts et al. (2015) found netic relationship of the genera Empis and Rhamphomyia. the genera to be polyphyletic, and that there is a Neotropi- cal linage of Empis more closely related to the tribe Hilar- ini Collin, 1961, sister to Empidini. It was also hypothe- Methods sized that further studies with additional sampling from the Palearctic and Nearctic regions and a larger molecular data Material set is necessary to resolve the phylogenetic relationships. Wahlberg and Johanson (2018) found Empis to be mono- A total of 48 taxa, 23 of Empis and 21 of Rhamphomy- phyletic except by two species of Rhamphomyia nested ia, representing twelve subgenera of Empis and nine within it, implying non-monophyly of Rhamphomyia, but of Rhamphomyia were sampled from the collections at far from all subgenera were represented in the study and the Swedish Museum of Natural History in Stockholm the specimens were mainly representatives of the Palearc- (NHRS). The subgenera of Empis sampled are Xan- tic Region. The genera Empis and Rhamphomyia have sev- thempis Bezzi, 1909, Kritempis Collin, 1926, Empis s. eral morphological resemblances involving a small head str., Euempis Frey, 1953, Anacrostichus Bezzi, 1909, with large eyes, elongated mouth parts, long legs and an Platyptera Meigen, 1803, Coptophlebia, Lissempis Bez- elongated abdomen with a high interspecific variability in zi, 1909, Polyblepharis Bezzi, 1909, Leptempis Collin, male genitalia. The morphology of females has been much 1926, Pachymeria Stephens, 1829, Planempis Frey,1953; less studied compared to that of the males, and identifica- and for Rhamphomyia are; Aclonempis, Amydroneura tion keys generally rely on male characters (Chvála 1994). Collin, 1926, Collinaria Frey, 1950, Eorhamphomyia Bothgenera can be morphologically distinguished from Frey, 1950, Holoclera Schiner, 1860, Lundstroemiella each other by the possession of a forked vein R4+5 in the Frey, 1922, Megacyttarus Bigot, 1880, Pararhamphomy- wings in Empis, a feature lacking in Rhamphomyia. In ad- ia Frey, 1922 and Rhamphomyia Meigen, 1822. Most of dition, Empis species have much longer mouthparts com- the samples were kept in 80% ethanol and were collect- pared to Rhamphomyia (Chvála 1994; Watts et al. 2016). ed in Sweden through the Swedish Malaise Trap Project Former studies on the phylogeny of Empis and Rham- (SMTP) (Karlsson et al. 2005) between 2003 and 2006. phomyia focused on either morphological or molecular Nine pinned specimens from 1843 to 1993 were included data from traditional Sanger sequencing on only few ge- from the dry collection of the NHRS. Unidentified spec- netic markers (Chvála 1994; Watts et al. 2016; Wahlberg imens were determined using the keys in Chvála (1994) and Johanson 2018. In this study we use a high through- and Collin (1961). Determined species were validated us- put sequencing (HTS) method named target enrichment. ing COI barcodes where reference data was available in This method allows for hundreds of genetic markers to be The Barcode of Life Data Systems (BOLD) (Ratnasing- analysed at a less time and money expense per marker and ham and Hebert 2007). Four specimens from three genera specimen compared to the traditional Sanger sequencing. from two tribes within Empididae were chosen as out- The targeted genetic markers focused on herein are called group; Hilarini (Hilara cornicula Loew, 1873, H. flavipes UCEs, ultraconserved elements. Which has proven useful Meigen, 1822) and Chelipodini Hendel, 1936 (Chelipoda for resolving phylogenies of less distant taxa of insects sp. Macquart, 1823 and Phyllodromia melanocephala (Faircloth et al. 2012; Faircloth et al. 2014; Blaimer et (Fabricius, 1794). The voucher numbers, collection data, al. 2016; Ješovnic et al. 2017; Van Dam et al. 2017). An- and author for each species, are listed in Table 1. other advantage of using this method is that it is useful on old and dried specimens which otherwise can pose a problem when using traditional Sanger sequencing due to DNA extraction and COI barcode amplification the fragmentation of old DNA (Blaimer et al. 2016). For this purpose, commercially synthesized bait set which is For DNA extraction the KingFisher™ Cell and Tissue complementary to the targeted UCEs sequences can be DNA Kit (Thermo Scientific, USA) was used together used to collect UCE data and its highly variable flanking with KingFisher™ Duo (Thermo Scientific, USA) ex- regions adjacent to the UCE loci. (Faircloth et al. 2012). traction robot following the manufacturer’s protocols. For Until this point UCEs have not yet been tested on dipter- extractions of large specimens one leg was removed from ans, this study is the first to evaluate the application of the body, for medium sized specimens the abdomen was UCEs on Diptera (Ultraconserved 2017). Amplification removed and for small ones the whole animal was used. of the cytochrome oxidase subunit I (COI) barcode gene Lysis was performed in 56 °C overnight. After extraction is also performed in order to evaluate if the extractions the body part was returned to the specimen. For pinned evolsyst.pensoft.net Evolutionary Systematics 4 2020, 21–33 23 material only oneg was used, due to restrictions from the amp.P5 forward library primer (10 μM) and IS6_reamp. museum. All extracted material is kept in 80% ethanol P7 reverse library primer (10 μM). After hybridization the as vouchers at the Swedish Museum of Natural History 8 pools were pooled into one pool in equimolar amounts (NHRS).